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Tracy L. Purcell, Yinhong Qu, Maria Eugenia Vola, Karin E. Thomas, Anne-Catherine Roch-Levecq, Josef F. Bille, David J. Schanzlin; Preliminary Safety Studies on Eye Bank Corneal Buttons Exposed to 2-Photon Ophthalmoscope Prototype. Invest. Ophthalmol. Vis. Sci. 2012;53(14):131.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the safety of examining live human cornea in vivo with the 2-photon ophthalmoscope by initial exposure of human eye bank corneal buttons.
Specular microscopy and vital staining (Trypan Blue/Alizarin Red) were used to determine cell loss on corneal tissue after exposure to a two-photon ophthalmoscope within laser safety limits.Laser Safety Calculation:According to Delori et al. (2007) and ANSI laser safety guidelines (Maximum permissible exposures for ocular safety, 2000), with emphasis on ophthalmic devices, with our system settings (central wavelength =780 nm, pulse width=150 fs, repetition rate =78 MHz, numerical Aperture= 0.8, irradiated area= 1536µm * 1536µm =0.0236cm2), the Maximum Permissible corneal irradiance is 94.4mW for long radiation (t>10s) and for shorter radiation the power limit for 6 frames (1.2s) is 515mW.Specular Microscopy and Vital Staining:Seven normal corneal buttons were prepared by the San Diego Eye Bank then placed in artificial anterior chambers. Five corneas were exposed on stage under the 2-photon scope (Exposed) and 2 buttons were placed on a table at room temperature (Controls). Laser power was set at a high level for Exposed buttons, 170mW. Exposure time ranged from 10 seconds to 1 hour and depth of tissue ranged from 0 (corneal surface) to 300 microns. Cell loss was determined by specular microcopy changes as well as final Trypan Blue/Alizarin Red staining using the Adobe Photoshop Method (Saad, et al., 2008).
2-photon laser Exposed corneas had cell loss range between 0% and 30.8%, depending on the technique used (specular microscopy vs vital staining, respectively), while Controls had loss of 4.26% to 30.0 %, respectively. Maximum loss between both groups was comparable. Vital staining in both groups was minimal and occurred mostly on the periphery of the cut buttons, while sparing the central endothelium.
The results of these findings indicate that the 170 mW 2-photon Exposed buttons were comparable to the Control buttons, demonstrating minimal endothelial cell loss, even up to 1 hour exposure and at 300 microns depth. Tested on more observations, this prototype may provide the possibility to non-invasively examine eyes in vivo, thereby allowing earlier detection and diagnosis of corneal diseases.
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